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  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/16—Amides, e.g. hydroxamic acids
  • A61K31/17—Amides, e.g. hydroxamic acids having the group >N—C(O)—N< or >N—C(S)—N<, e.g. urea, thiourea, carmustine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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  • A61P17/00—Drugs for dermatological disorders
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• • A—HUMAN NECESSITIES
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  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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• • A—HUMAN NECESSITIES
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  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
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Definitions

• This invention relates to methods of inhibiting leukotriene biosynthesis. In another aspect this invention relates to methods of treating diseases mediated by leukotrienes. In yet another aspect this invention relates to pharmaceutical compositions for inhibiting leukotriene biosynthesis. Description of the Related Art
• Arachidonic acid a component of phospholipids found in cell membranes, is metabolized through an array of enzymatic pathways to afford biologically active metabolites including the leukotrienes.
• the leukotrienes are very potent substances, producing a variety of biological effects when present in the nanomolar or picomolar concentration range. They have been implicated in a variety of disease states.
• Leukotriene C 4 and Leukotriene D 4 are potent constrictors of human airway smooth muscle. Aerosol administration of these substances to nonasthmatic volunteers induces bronchoconstriction.
• Leukotriene B 4 is a potent chemotactic factor for inflammatory cells such as polymorphonuclear leukocytes.
• Leukotriene B 4 has been found in the synovial fluids of rheumatoid arthritis patients and in psoriatic lesions. Leukotrienes have also been implicated as important mediators in allergic rhinitis, adult respiratory distress syndrome, inflammatory bowel disease, ischemic induced myocardial injury, reperfusion injury, gout, asthma, psoriasis, stroke, spinal cord injury and traumatic brain injury.
• This invention provides a method of inhibiting leukotriene biosynthesis in an animal and of treating in an animal a condition responsive to such inhibition, comprising administering to the animal, in an amount effective to inhibit leukotriene biosynthesis, a compound of Formula l:
• R is selected from the group consisting of hydrogen; cyclic alkyl containing five to ten carbon atoms; straight chain or branched chain alkyl containing one to fourteen carbon atoms and substituted straight chain or branched chain alkyl containing one to twelve carbon atoms, wherein the substituent is alkoxycarbonyl wherein the alkoxy group contains one to four carbon atoms; alkoxyalkyl wherein the alkoxy moiety contains one to six carbon atoms and the alkyl moiety contains one to six atoms;
• each R' is independently selected from the group consisting of halogen; nitro; straight chain or branched chain alkyl containing one to five carbon atoms; alkoxy containing one to four carbon atoms; alkoxyphenyl wherein the alkoxy group contains one to eight carbon atoms; alkylthio containing one to four carbon atoms; alkylsulfonoxy containing one to four carbon atoms; alkylsulfinyl containing one to four carbon atoms; alkylsulfonyl containing one to four carbon atoms;
• R" is selected from the group consisting of hydrogen and straight chain alkyl containing one to twelve carbon atoms
• M is selected from the group consisting of hydrogen, a pharmaceutically acceptable cation, and a pharmaceutically acceptable metabolically cleavable group with the proviso that R, R', and R" are not all hydrogen.
• This invention also provides a pharmaceutical composition for the treatment of an animal having a condition responsive to treatment by inhibition of leukotriene biosynthesis, comprising (i) a pharmaceutically acceptable vehicle, and (ii) a compound of Formula I where n, R, R', R", and M are as defined above in an amount effective to inhibit leukotriene biosynthesis.
• This invention also provides for the use of a compound of Formula I in the manufacture of a pharmaceutical composition for use in inhibiting leukotriene biosynthesis.
• the method of this invention comprises administering a compound of Formula I:
• R substituent is defined above.
• Preferred R substituents include straight chain or branched chain alkyl containing one to six carbon atoms and cycloalkyl containing five to eight carbon atoms.
• R is alkyl as defined above, preferred R substituents include methyl, 1-methylethyl, 1-ethylpropyl, and 1-methylpropyl.
• R' substituents include halogen, nitro, straight chain alkyl containing one to four carbon atoms, alkylthio containing one to four carbon atoms and phenoxy.
• R' is alkylthio as defined above, the preferred R' substituent is thiomethyl.
• Preferred R" substituents include hydrogen and methyl.
• n is 0, 1, 2 or 3, preferably 0 or 1.
• R' may be in the ortho, meta or para position, preferably the para position, on the phenyl ring.
• the M substituent is selected from the group consisting of hydrogen, a pharmaceutically acceptable cation, and a pharmaceutically acceptable metabolically cleavable group.
• pharmaceutically acceptable cation refers to nontoxic cations well known to those skilled in the art and including but not limited to those based on the alkali and alkaline earth metals such as sodium, lithium, potassium, magnesium, aluminum and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, derived from nitrogenous bases of sufficient basicity to form salts with the N-hydroxy group of the compounds of Formula I (where M is hydrogen).
• Metabolically cleavable group denotes a moiety that is readily cleaved in vivo from the compound bearing it. The compound after cleavage remains or becomes pharmacologically active. Metabolically cleavable groups generally are derived from compounds (well known to those skilled in the art) reactive with the N-hydroxy group of compound of Formula I where M is hydrogen.
• Such groups include, but are not limited to, alkanoyl (such as acetyl, propionyl and the like), unsubstituted and substituted aroyl (such as benzoyl and substituted benzoyl), alkoxycarbonyl (such as ethoxycarbonyl), monoesters formed with dicarboxylic acids (such as succinyl), and the like.
• alkanoyl such as acetyl, propionyl and the like
• unsubstituted and substituted aroyl such as benzoyl and substituted benzoyl
• alkoxycarbonyl such as ethoxycarbonyl
• monoesters formed with dicarboxylic acids such as succinyl
• M is preferably hydrogen.
• Preferred compounds for use in the method of this invention include:
• Reaction Scheme I involves reacting a hydroxylamine of Formula II with an aryl isocyanate of Formula III to afford a hydroxyurea of Formula IV.
• Formula IV is a subgenus of Formula I.
• Many hydroxylamines of Formula II are commercially available. Others may be readily prepared using conventional methods, for example, conversion of a suitable ketone to its oxime followed by reduction to the requisite hydroxylamine. Many isocyanates of Formula III are also commercially available. Others may be readily prepared using conventional methods, for example, Curtius rearrangement, Hofmann rearrangement, Schmidt reaction, or reacting an aniline with phosgene.
• the reaction in Reaction Scheme I can be conducted at ambient temperature by dropwise addition of the isocyanate to a solution of the
• a suitable solvent e.g., an aprotic solvent such as diethyl ether or tetrahydrofuran.
• a salt e.g., a hydrochloride
• it is converted to the free base using conventional means (e.g., reacting with one equivalent of base in a suitable solvent) prior to its reaction with the isocyanate.
• Reaction Scheme II Certain compounds of Formula I can also be prepared according to Reaction Scheme II, wherein n, R and R' are as defined above. Reaction Scheme II, however, is generally not suitable for compounds in which R' is a strongly electron withdrawing group (e.g., nitro).
• R' is a strongly electron withdrawing group (e.g., nitro).
• step (1) of Reaction Scheme II O-benzylhydroxylamine hydrochloride is reacted, according to the method of Reaction Scheme I, with an isocyanate of Formula III to afford a 1-benzyloxy-3 -phenylurea of Formula V.
• step (2) the compound of Formula V is alkylated under conventional conditions, e.g., by reacting with a compound of formula RBr in the presence of a base such as sodium hydride and in a suitable polar aprotic solvent (e.g., N,N-dimethylformamide) to afford a benzyloxy urea of Formula VI.
• a base such as sodium hydride
• a suitable polar aprotic solvent e.g., N,N-dimethylformamide
• step (3) of Reaction Scheme II the benzyloxy urea of Formula VI is debenzylated using conventional means (e.g., hydrogenolysis) to afford the hydroxy urea of Formula IV .
• step (1) of Reaction Scheme III O-benzylhydroxylamine hydrochloride is reacted with di-tert-butyldicarbonate to provide tert-butyl N-benzyloxycarbamate.
• step (2) tert-butyl benzyloxycarbamate is alkylated under conventional conditions, e.g., by reacting with a compound of formula RBr in the presence of a base such as sodium hydride and in a suitable polar aprotic solvent (e.g., N,N- dimethylformamide). The alkylation affords a carbamate of Formula VII. The reaction can be heated if necessary or desirable.
• a base such as sodium hydride
• a suitable polar aprotic solvent e.g., N,N- dimethylformamide
• step (3) of Reaction Scheme III the carbamate of Formula VII is deblocked by treating with trifluoroacetic acid in a suitable solvent (e.g., methylene chloride) to afford an O-benzylhydroxylamine of Formula VIII.
• a suitable solvent e.g., methylene chloride
• step (4) the O- benzylhydroxylamine of Formula VIII is reacted according to the method of Reaction Scheme I with an isocyanate of Formula III to afford a benzyloxy urea of Formula VI.
• step (5) the benzyloxy urea is debenzylated using conventional means (e.g., hydrogenolysis) to afford a hydroxy urea of Formula IV.
• step (1) of Reaction Scheme IV an O-benzylhydroxylamine of Formula VIII is reacted with a carbamoyl chloride of Formula IX to afford a benzyloxy urea of Formula X.
• O-benzylhydroxylamines of Formula VIII may be prepared according to the method of Reaction Scheme III.
• Many carbamoyl chlorides of Formula IX are commercially available. Others may be readily prepared using conventional methods, for example, reacting an N-alkylaniline with phosgene
• the reaction in step (1) can be carried out in a sealed tube at an elevated temperature in the presence of a base (e.g., triethylamine) in a suitable solvent (e.g., an aprotic solvent such as tetrahydrofuran).
• step (2) of Reaction Scheme IV the benzyloxy urea of Formula X is debenzylated using conventional means (e.g., hydrogenolysis) to afford the hydroxyurea of Formula XI.
• Certain compounds of Formula I can also be prepared according to Reaction Scheme V, wherein n, R, R' and R" are as defined above, provided however that R" is not hydrogen.
• step (1) of Reaction Scheme V O-benzylhydroxylamine hydrochloride is reacted with a carbamoyl chloride of Formula IX to afford a benzyloxy urea of Formula XII.
• the reaction can be conducted at ambient temperature by combining the reactants in a suitable solvent (e.g., diethyl ether).
• the O-benzylhydroxylamine hydrochloride is converted to the free base using conventional means (e.g., reacting with one equivalent of base in a suitable solvent) prior to its reaction with the carbamoyl chloride.
• step (2) of Reaction Scheme V the compound of Formula XII is alkylated under conventional conditions, e.g., by reacting with an alkyl halide in the presence of a base such as sodium hydride and in a suitable polar aprotic solvent (e.g., N,N-dimethylformamide) to afford a benzyloxy urea of Formula X.
• a base such as sodium hydride
• a suitable polar aprotic solvent e.g., N,N-dimethylformamide
• step (3) of Reaction Scheme V the benzyloxy urea of Formula X is debenzylated using conventional means (e.g., hydrogenolysis) to afford the hydroxyurea of Formula XI.
• Certain compounds of Formula I can be prepared by elaboration of the R' group by conventional means (e.g., oxidation of an alkylthio group to an alkyl sulfonyl group) at an appropriate stage of synthesis
• the compounds of Formula I wherein M is a pharmaceutically acceptable cation can be prepared by combining in a polar solvent a compound of Formula I where M is hydrogen with an equimolar amount of a relatively strong base, e.g., a base of the formula M(OH) x wherein M is the pharmaceutically acceptable cation and x is the valence of such cation. Isolation of the salt can be facilitated by the addition of a solvent, such as diethyl ether, in which the salt is insoluble.
• a compound of Formula I can be formulated for various routes of administration (e.g., oral administration by tablet, capsule, oral suspension, or the like) in an appropriate pharmaceutically acceptable vehicle and adjuvants and excipients suitable for the selected dosage form.
• a solid dosage form for example, contains a compound of Formula I, and one or more ingredients selected from the group consisting of diluents (e.g., dicalcium phosphate, calcium sulfate, lactose, mannitol, cellulose, kaolin, sodium chloride, starch, sucrose, inositol, sorbitol), binders (e.g., starch, gelatin, sucrose, glucose, dextrose, molasses, lactose, natural and synthetic gums), lubricants (e.g., talc, magnesium stearate, calcium stearate, stearic acid,
• diluents e.g., dicalcium phosphate, calcium sulfate, lactose, mannitol, cellulose, kaolin, sodium chloride, starch, sucrose, inositol, sorbitol
• binders e.g., starch, gelatin, sucrose
• the compound of Formula I is administered in an amount effective to inhibit leukotriene biosynthesis.
• the amount that constitutes an amount effective to inhibit leukotriene biosynthesis will depend on the particular compound, the particular formulation, the route of administration, and the intended therapeutic effect. Those skilled in the art can determine such an amount with due consideration of such factors.
• a number of the compounds of Formula I are shown by the Examples below to inhibit leukotriene biosynthesis. These results suggest utility in treating conditions mediated by leukotrienes, such as arthritis, rheumatoid arthritis, osteoarthritis, allergic rhinitis, adult respiratory distress syndrome, inflammatory bowel disease, ischemic induced myocardial injury, reperfusion injury, gout, asthma, stroke, psoriasis, spinal cord injury, and traumatic brain injury. in the following syntheses, the structures of the final compounds (i.e.
• N-methylhydroxylamine hydrochloride was reacted with an isocyanate of Formula III to provide the compounds of Formula I shown in Table 1.
• the melting points and elemental analyses are shown in Table 2.
• N-Isopropylhydroxylamine hydrochloride (1.35 g, 12.1 mmol) was dissolved in water (2 mL) then combined with diethyl ether (25 mL) followed by the addition of a solution of sodium hydroxide (0.48 g, 12.1 mmol) in water (2 mL). The reaction mixture was allowed to stir for several minutes then 4-(methylthio)- phenyl isocyanate (2.0 g, 12.1 mmol) was added via pipette. A white precipitate formed. The reaction mixture was allowed to stir at ambient temperature overnight. The solid was isolated by filtration then dissolved in hot ethyl acetate The ethyl acetate solution was dried over magnesium sulfate then concentrated under vacuum. The precipitate was collected and dried to afford 1.60 g of the desired product as a crystalline solid, m.p. 150.0-150.3°C. Analysis: Calculated for
• N-cyclohexylhydroxylamine hydrochloride (1.5 g, 10 mmol) was reacted with 4-(methylthio)phenyl isocyanate
• the reaction was quenched with water then extracted with several portions of diethyl ether.
• the ether extracts were combined, washed with water and brine, dried over magnesium sulfate and then concentrated to provide 8.7 g of crude product as a pale yellow solid.
• the crude product was purified by flash chromatography (silica gel; 9:1 hexanes: ethyl acetate) to provide 5.7 g of 1 -benzyloxy- 1-(1 -ethylpropyl)-3- phenylurea as a white solid, m.p. 45.0-45.6°C.
• Trifluoroacetic acid (21 mL, 270 mmol) was added in a single portion to a solution of tert-butyl N-benzyloxy-N-(2-methylpropyl)carbamate (7.6 g, 27 mmol) in methylene chloride (21 mL). A spontaneous reflux occurred.
• the reaction mixture was stirred without external heating for 20 minutes and then concentrated under vacuum at 45°C.
• Saturated aqueous sodium bicarbonate was added to the residue followed by a quantity of solid sodium bicarbonate sufficient to bring the pH of the reaction mixture up to a value of 9.
• the mixture was extracted twice with methylene chloride. The extracts were combined, dried over magnesium sulfate then concentrated under vacuum to provide 4.9 g of N-benzyloxy-N-(2- methylpropyl)amine as an oil.
• Phenyl isocyanate (3.2 g, 27 mmol) was added via pipette to a solution of N-benyloxy-N-(2-methylpropyl)amine (4.8 g, 27 mmol) in methylene chloride (30 mL). The reaction mixture was allowed to stir at ambient temperature for 3 days. The solvent was removed under vacuum. The residue was purified by column chromatography (silica gel; 1 :9 ethyl acetate: hexanes) to provide 6.2 g of 1- benzyloxy-1-(2-methylpropyl)-3-phenylurea as a white crystalline solid, m.p. 85- 87°C. Analysis: Calculated for C 18 H 22 N 2 O 2 : %C, 72.46; %H, 7.43; %N, 9.39, Found: %C, 72.53; %H, 7.51; %N, 9.44.
• Diethyl ether (10 mL) was added to a suspension of O-benzylhydroxylamine hydrochloride (0.82 g, 5.1 mmol) in water (1 mL).
• a solution of sodium hydroxide (0.23 g, 5.8 mmol) in water (1 mL) was added and the mixture was stirred for several minutes.
• the aqueous layer was removed by pipette and extracted with diethyl ether (3 mL).
• the ether extract was added to the reaction flask.
• 4- Butylphenyl isocyanate (0.90 g, 5.1 mole) was added to the reaction flask via pipette in a single portion. A mild reflux occurred.
• Trifluoroacetic acid (42 mL, 544 mmole) was added to a chilled (0°C) solution of tert-butyl N-benzyloxy-N-(1-ethylpropyl)carbamate (21.6 g, 73.6 mmole) in methylene chloride (300 mL). The reaction was stirred at ambient temperature for about 18 hours then concentrated under vacuum. Saturated aqueous sodium bicarbonate (100 mL) was added to the residue followed by a quantity of solid sodium carbonate sufficient to adjust the pH of the reaction mixture to pH 9. The reaction mixture was extracted with diethyl ether (4 X 50 mL). The extracts were combined, dried over sodium sulfate then concentrated to provide 10.9 g of N-benzyloxy-N-(1-ethylpropyl)amine as an oil.
• O-Benzylhydroxylamine hydrochloride (3.0 g, 18.8 mmole) was dissolved in water (3.5 mL) then combined with diethyl ether (85 mL) followed by the addition of sodium hydroxide (0.75 g, 18.8 mmole) in water. After several minutes a solution of N-methyl-N-phenylcarbamoyl chloride (3.19 g, 18.8 mmole) in diethyl ether (25 mL) was added. A white precipitate formed. The solid was isolated by filtration then dissolved in water. The solution was diluted with saturated aqueous sodium bicarbonate and sufficient solid sodium carbonate was added to bring the pH to 9.
• the solution was extracted with diethyl ether (5 X 50 mL).
• the ether extracts were combined, dried and then concentrated to provide the crude product as a beige solid.
• the crude product was purified by silica gel chromatography (9: 1 hexanes/ethyl acetate) to provide 3.1 g of 1-benzyloxy-3-methyl-3-phenylurea.
• methyl 4- hydroxybenzoate (3.0 g, 19.7 mmole) was reacted with 1-bromo-3-phenylpropane (3.1 mL, 20.4 mmole) to provide 4.8 g of methyl 4-(3-phenylpropyloxy)benzoate as a white crystalline solid, m.p. 61.0-61.5°C.
• Potassium carbonate (27.6 g, 0.20 mole) was added to a solution of cyclopentyl bromide (26.4 g, 0.18 mole) and isovanillin (25.8 g, 0.17 mole) in N,N- dimethyl formamide (80 mL). The resulting suspension was stirred at ambient temperature for 1 week, then diluted with water (about 500 mL) and extracted with diethyl ether (2 X 300 mL).
• test method described below measures the ability of compounds to inhibit 5-lipoxygenase activity in human leukocytes
• the red blood cells are sedimented with a 6% dextran/0.9% sodium chloride solution at a ratio of 25 mL whole blood to 15 mL dextran solution.
• the blood/dextran combination is mixed by inversion and the red blood cells are allowed to settle out for 45 minutes at ambient temperature.
• the plasma/dextran supernatant is removed then centrifuged at ambient temperature at 3000 rpm for 10 minutes.
• the plasma/dextran supernatant is removed and the cells are resuspended in 10 mL of the plasma/dextran solution.
• the cell suspension is combined with 20 mL of water, mixed for 1.5 minutes then immediately combined with 10 mL of 3.6% sodium chloride, mixed and centrifuged at ambient temperature at 3000 rp m for 10 minutes.
• the pellet is washed with 40 mL of Tris buffer (5.55 mM dextrose, 15.36 mM Tris base, 136.9 mM sodium chloride with pH 7.3-7.4) then centrifuged at 3000 rp m for 10 minutes.
• the pellet is then resuspended into Tris buffer containing 1 mM calcium chloride to provide approximately 1.0 X 10 7 cells/mL.
• Compounds are dissolved in dimethyl sulfoxide. Compounds are tested at concentrations of 100, 33, 11, 3.7, 1.2 and 0.41 ⁇ M. Each concentration is tested in duplicate.
• a 15 ⁇ L portion of Tris buffer containing 1 mM calcium chloride is added to each well of a 96 well microtiter plate.
• a 1 ⁇ L portion of drug solution or vehicle (dimethyl sulfoxide) is added to each well followed by the addition of an 75 ⁇ L portion of the cell suspension.
• the plates are gently mixed then allowed to stand at ambient temperature for 10 minutes.
• a 10 ⁇ L portion of 30 ⁇ M A23187 Calcium Ionophore (prepared by dissolving the ionophore in DMSO and then diluting 1 :80 into the Tris buffer) is added to each well except the wells that contain the blanks (The blank wells measure the level of LTC 4 production in the absence of A23187.).
• the plates are gently mixed then incubated at 37°C for 10 minutes.
• the supernatants are assayed for the presence of Leukotriene C 4 by radioimmunoassay which is performed according to the manufacturer's (Advanced Magnetics; Cambridge, MA) instructions.
• Inhibition of 5-lipoxygenase activity is calculated as the ratio of the amount of LTC 4 formed in the presence (LTC 4 + cpd) and absence (LTC 4 no cpd) of compound according to the following equation.
• IC 50 values concentration of compound producing 50% enzyme inhibition are calculated by linear regression analysis of percentage inhibition versus log compound concentration plots.
• test method described below measures the ability of compounds to inhibit the production of Leukotriene B 4 in whole human blood.
• Compounds are dissolved in dimethyl sulfoxide. Compounds are tested at concentrations of 100, 33, 11, 3.7, 1.2 and 0.41 ⁇ M. Each concentration is tested in duplicate.
• the tubes are centrifuged at 2000 rp m for 10 minutes. 100 ⁇ L portions of plasma are transferred to 1 mL polyethylene tubes containing 400 ⁇ L portions of methanol. The tubes are vortexed then frozen at -20°C overnight. Analysis/Calculations
• the tubes are centrifuged for 10 minutes then 100 ⁇ L portions of methanol supernatant are transferred to a 96 well microtiter plate. 10 ⁇ L portions are transferred from this plate to a 96 well assay plate. Methanol dilutions of LTB 4 standard curve are added to the assay plate. 10 ⁇ L portions of blank
• LTB 4 production is calculated as the ratio of the amount of LTB 4 formed in the presence (LTB 4 + cpd) and absence (LTB 4 no cpd) of compound according the equation below.
• IC 50 values concentration of compound producing a 50% inhibition of LTB 4 production are calculated by linear regression analysis of percentage inhibition versus log compound concentration plots.
• test method described below measures the ability of compounds to inhibit the production of Leukotriene C 4 in mouse peritoneal macrophages.
• mice female, CD-1, weighing 25 g are euthanized by exposure to carbon dioxide.
• the peritoneal cavity is exposed by peeling back the abdominal skin.
• a 5 mL portion of media (M199 containing 1% fetal bovine serum, 100 units/mL of penicillin, 100 ⁇ g/mL of streptomycin, 20 units/mL of heparin and no glutamine) is injected into the exposed peritoneal cavity of each mouse.
• the lavage fluid is removed and pooled to yield approximately 1 X 10 6 macrophages/mL.
• a 2 mL portion of lavage fluid is added to each well of a 24 well sterile multidish and the macrophages are allowed to adhere to the plate for 2 hours at 37°C in a 5% carbon dioxide atmosphere.
• the media is removed and each well is washed with 2 mL of phosphate buffered saline (PBS).
• PBS phosphate buffered saline
• a 1 mL portion of media, without heparin, but containing 5 ⁇ Ci/mL of 3H-myoinositol is added to each well and the plates are incubated overnight at 37°C in a 5% carbon dioxide atmosphere.
• the media is removed and the cells are washed twice with 2 mL portions of PBS.
• Puck's saline formulation A containing 1 mM calcium chloride, 1 mM magnesium chloride and 10 mM lithium chloride is added to each well.
• the Puck's formulation is made first as a 10X solution which contains 4 g of potassium chloride, 80 g of sodium chloride and 10 g of glucose per liter.
• the Puck's saline formulation A is made using 10 mL of the 10X Puck's formulation, 0.47 mL of 7.5% sodium bicarbonate and 2 mL of 1M N-2-hydroxyethylpiperazine-N-2- ethanesulfonic acid per 100 mL.
• Compounds are dissolved in dimethyl sulfoxide. Compounds are tested at concentrations of 10, 1 and 0.1 ⁇ M. Each concentration is tested in duplicate. Incubation
• a 1 ⁇ L portion of compound solution or vehicle (DMSO) is added to each well and the plates are incubated for 15 minutes at 37°C in a 5% carbon dioxide atmosphere.
• Zymosan is then added to provide a final concentration of 50 ⁇ g/mL in each well and the plate is incubated for 1 to 2 hours at 37°C in a 5% carbon dioxide atmosphere.
• the media is assayed for the presence of Leukotriene C 4 by
• the test method described below measures the ability of a compound when administered orally to rats to inhibit the production of Leukotriene B 4 in their blood which is drawn and challenged.
• Rats (CD, male, non-fasted, 250 g) are lightly anesthetized with carbon dioxide and an approximately 0.75 mL sample of whole blood is obtained via cardiac puncture.
• the sample is dispensed into 12 X 75 mm polypropylene tubes containing 8-10 ⁇ L of 10,000 units/mL heparin, mixed and then maintained on ice.
• the rats are allowed to recover approximately one hour then dosed orally with compound dissolved in PEG 400 at a 5 mL/Kg volume. Five (5) rats are utilized per group. Two (2) hours post dose the rats are again anesthetized with carbon dioxide and the blood sampled again via cardiac puncture.
• Duplicate 200 ⁇ L portions of blood are added to 1.0 mL polypropylene tubes.
• a 10 ⁇ L portion of 1mM A23187 Calcium Ionophore in dimethyl sulfoxide/Tris buffer is added to each tube.
• the tubes are gently vortexed then incubated in a 37°C water bath for 30 minutes.
• the tubes are then centrifuged at 4000 rp m for 10 minutes.
• 50 ⁇ L portions of plasma are transferred to 1.0 mL tubes containing 200 ⁇ L of methanol.
• the tubes are vortexed then placed in the freezer overnight.
• the tubes are removed from the freezer then centrifuged at 4000 rp m for 10 minutes. 20 ⁇ L portions of the methanol/plasma supernatant and 10 ⁇ L methanol dilutions of LTB 4 standard curve are transferred to 96 well v-bottom microtiter plates. The plates are vacuum dried at 40°C. A 40 ⁇ L portion of LTB 4
• radioimmunoassay buffer is added to each well.
• the plate is bath sonicated for 5 minutes then assayed according to the manufacturer's (Advanced Magnetics;
• DOG EX VIVO LEUKOTRIENE B 4 INHIBITION The test method described below measures the ability of a compound when administered orally to dogs to inhibit the production of Leukotriene B 4 (LTB 4 ) in their blood which is drawn and challenged at selected time points.
• the test is conducted in beagle dogs (male and female; about 10 Kg). Prior to starting, the neck area of the beagle is shaved to allow easy access to the jugular vein.
• a 3-4 mL sample of blood is drawn from the jugular vein into a heparinized vacutainer. The dog is then dosed using a tube long enough to reach its stomach. The compound is dissolved in PEG 400 at a concentration such that the appropriate dose can be administered in approximately 5 mL. Immediately after the compound is given, a flush using 5 mL of vehicle is given to rinse the dosing tube. At selected time points following the dose, a 3-4 mL sample of blood is drawn.
• the blood samples that have been drawn are challenged as soon as possible.
• a 0.5 mL portion of blood is aliquoted into 3 tubes (1 mL microtiter tubes).
• a 1 ⁇ L portion of 25 mM calcium ionophore in dimethyl sulfoxide is added to 2 of the tubes.
• a 1 ⁇ L portion of dimethyl sulfoxide is added to the third tube.
• the tubes are lightly mixed then placed in a 37°C water bath for 30 minutes
• the tubes are centrifuged at 11,000 ⁇ m for 3 minutes and the plasma supernatant is transfe ⁇ ed to clean tubes.
• a 50 ⁇ L portion of this plasma is added to a 1 mL microtiter tube containing 200 ⁇ L of methanol.
• the tubes are placed in the freezer at -20°C for at least 1 hour then centrifuged at 2000 ⁇ m for 10 minutes. A 10 ⁇ L portion of the methanol supernatant is dried under vacuum at ambient temperature and then reconstituted in LTB 4 buffer. The amount of LTB 4 in the sample is determined by radioimmunoassay performed according to the manufacturer's (Advanced

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• 1995
  • 1995-07-14 AU AU30917/95A patent/AU685980B2/en not_active Ceased
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• 1997
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